This invention relates to a method of using elongated, needle-like microfibers or xe2x80x9cwhiskersxe2x80x9d to transform plant cell aggregates and selected plant tissues.
Until recently, genetically manipulated plants were limited almost exclusively to those events created by application of classical breeding methods. Creation of new plant varieties by breeding was reserved primarily for the most agronomically important crops, such as corn, due to the cost and time needed to identify, cross, and stably fix a gene in the genome, thus creating the desired trait. In comparison, the advent of genetic engineering has resulted in the introduction of many different heterologous genes and subsequent traits into diverse crops including corn, cotton, soybeans, wheat, rice, sunflowers and canola in a more rapid manner. However, the intergression of a new transgene into elite germplasm is still quite a laborious task due to the tissue culturing and back-crossing needed to produce a commercially viable, elite, line.
Several techniques exist which allow for the introduction, plant regeneration, stable integration, and expression of foreign recombinant vectors containing heterologous genes of interest in plant cells. One such technique involves acceleration of microparticles coated with genetic material directly into plant cells (U.S. Pat. No. 4,945,050 to Cornell; U.S. Pat. No. 5,141,131 to DowElanco; and U.S. Pat. Nos. 5,538,877 and 5,538,880, both to Dekalb). This technique is commonly referred to as xe2x80x9cmicroparticle bombardmentxe2x80x9d or xe2x80x9cbiolisticsxe2x80x9d. Plants may also be transformed using Agrobacterium technology (U.S. Pat. No. 5,177,010 to University of Toledo, U.S. Pat. No. 5,104,310 to Texas AandM, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500 all to Max Planck, European Patent Applications 604662,627752 and U.S. Pat. No. 5,591,616 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba-Geigy, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135 both to Agracetus). Another transformation method involves the use of elongated needle-like microfibers or xe2x80x9cwhiskersxe2x80x9d to transform cell suspension cultures (U.S. Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca). In addition, electroporation technology has been used to transform plant cells from which fertile plants have been obtained (WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and 5,384,253 both to Dekalb; U.S. Pat. No. 5,679,558, 5,641,664, and WO9209696 and WO9321335 to Plant Genetic Systems).
Despite all of the technical achievements, genetic transformation and routine production of transgenic plants in a commercially viable, elite, germplasm is still a laborious task. For example, microparticle bombardment, while capable of being used either on individual cells, cell aggregates, or plant tissues, requires preparing DNA-attached gold particles and optimization of an expensive and not yet widely available, xe2x80x9cgunxe2x80x9d apparatus. Techniques involving Agrobacterium are extremely limited because not all plant species or varieties within a given species are susceptible to infection by the bacterium. Electroporation techniques are not preferred due to the extreme difficulties and cost typically encountered in routinely making protoplast from different plant species and tissues thereof and the concomitant low viability and low transformation rate associated therewith.
As disclosed herein, applicants have invented a method whereby plant cell aggregates and plant tissues from non-elite and elite germplasm can be directly and inexpensively transformed with a recombinant vector containing the gene of choice using whiskers. Applicants"" invention is advantageous over currently used methods in that it is simple, quick and easy to use. Furthermore, applicants"" invention is superior to that described in the art in that it eliminates the need to establish Type III callus cultures or establish and maintain cell suspension cultures, and can be used with either Type I or Type II callus, thus, is less germplasm limited. This means that applicants"" invention, as described herein, can be used to transform elite genotypes directly thus eliminating the problems and time generally associated with gene intergression.
The present invention relates to the production of fertile, transgenic, Zea mays plants containing heterologous DNA preferably integrated into the chromosome of said plant and heritable by the progeny thereof.
One aspect of the present invention relates to Zea mays plants, plant parts, plant cells, plant cell aggregates, and seed derived from transgenic plants containing said heterologous DNA.
The present invention also relates to the production of fertile, transgenic, Oryza sativa L. plants containing heterologous DNA preferably integrated into the chromosome of said plant and heritable by the progeny thereof.
Another aspect of the present invention relates to Oryza sativa L. plants, plant parts, plant cells, plant cell aggregates, and seed derived from transgenic plants containing said heterologous DNA.
The present invention also relates to the production of fertile, transgenic, Gossypium hirsutum L. plants containing heterologous DNA preferably integrated into the chromosome of said plant and heritable by the progeny thereof.
Another aspect of the present invention relates to Gossypium hirsutum L. plants, plant parts, plant fibers, plant cells, plant cell aggregates, and seed derived from transgenic plants containing said heterologous DNA.
The invention further relates to a process for producing fertile transformed plants from Type I callus, Type II callus, hypocotyl-derived callus, or cotyledon-derived callus by whisker-mediated transformation.
The invention yet further relates to a process for producing fertile transformed plants from meristematic tissue by whisker-mediated transformation.
Another aspect of the invention relates to fertile, mature maize plants regenerated from Type I or Type II callus and transgenic seed produced therefrom.
Another aspect of the invention relates to fertile, mature rice plants regenerated from Type I callus and transgenic seed produced therefrom.
Yet, another aspect of the invention relates to fertile, mature cotton plants regenerated from hypocotyl-derived callus or cotyledon-derived callus and transgenic seed and fiber produced therefrom.
In a preferred embodiment, this invention produces the fertile transgenic plants described herein by means of whisker-mediated cell perforation and heterologous DNA uptake, said whisker-mediated cell perforation being performed on plant cell aggregates and plant tissues, followed by a controlled regimen for selection and production of transformed plant lines.
Other aspects, embodiments, advantages, and features of the present invention will become apparent from the following specification.
The present invention relates to methods for production of fertile transgenic plants and seeds of, for example, the species Zea mays, Oryza sativa L., Gossypium hirsutum L., and Brassica napus by transforming plant cell aggregates and plant tissues of said species with the DNA construct of interest via whisker-mediated transformation. After transformation, transgenic plants are regenerated from said transformed plant cell aggregates and plant tissues and said regenerated plants express the chimeric DNA construct of interest. The transgenic plants produced herein by the methods described include: all species of corn including but not limited to field corn, popcorn, sweet corn, flint corn, dent corn and the like; all species of cotton; and all species of rice.
The following phrases and terms are defined below:
By xe2x80x9cantisensexe2x80x9d is meant an RNA transcript that comprises sequences complementary to a target RNA and/or mRNA or portions thereof and that blocks the expression of a target gene by interfering with the processing, transport, and/or translation of its primary transcript and/or mRNA. The complementarity may exist with any part gof the target RNA, i.e., the 5xe2x80x2 non-coding sequence, 3xe2x80x2non-coding sequence, introns, or the coding sequence. Antisense RNA is typically a complement (mirror image) of the sense RNA.
By xe2x80x9ccDNAxe2x80x9d is meant DNA that is complementary to and derived from a mRNA.
By xe2x80x9cchimeric DNA constructionxe2x80x9d is meant a recombinant DNA containing genes or portions thereof from one or more species in either the sense or antisense orientation.
By xe2x80x9cconstitutive promoterxe2x80x9d is meant promoter elements that direct continuous gene expression in all cell types and at all times (i.e., actin, ubiquitin, CaMV 35S, 35T, and the like).
By xe2x80x9ccosuppressionxe2x80x9d is meant the introduction of a foreign gene having substantial homology to an endogenous gene, and in a plant cell causes the reduction in activity of the foreign gene and/or the endogenous gene product. Cosuppression can be sometimes achieved by introducing into said plant cell either the promoter sequence, the 5xe2x80x2 and/or 3xe2x80x2 ends, introns or the coding region of a gene.
By xe2x80x9cdevelopmental specificxe2x80x9d promoter is meant promoter elements responsible for gene expression at specific plant developmental stages, such as in early or late embryogenesis and the like.
By xe2x80x9cenhancerxe2x80x9d is meant nucleotide sequence elements which can stimulate promoter activity such as those from maize streak virus (MSV), alfalfa mosaic virus (AMV), alcohol dehydrogenase intron 1 and the like.
By xe2x80x9cexpressionxe2x80x9d as used herein, is meant the transcription of enzymatic nucleic acid molecules, mRNA, and/or the antisense RNA inside a plant cell. Expression of genes also involves transcription of the gene and may or may not involve translation of the mRNA into precursor or mature proteins.
By xe2x80x9cforeignxe2x80x9d or xe2x80x9cheterologous genexe2x80x9d is meant a gene having a DNA sequence that is not normally found in the host cell, but is introduced by whisker-mediated transformation.
By xe2x80x9cgenexe2x80x9d is meant to include all genetic material involved in protein expression including chimeric DNA constructions, genes, plant genes and portions thereof.
By xe2x80x9cgenomexe2x80x9d is meant genetic material contained in each cell of an organism and/or virus.
By xe2x80x9cinducible promoterxe2x80x9d is meant promoter elements which are responsible for expression of genes in response to a specific signal, such as: physical stimuli (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites, stress and the like.
By xe2x80x9cmodified plantxe2x80x9d is meant a plant wherein the mRNA levels, protein levels or enzyme specific activity of a particular protein have been altered relative to that seen in an unmodified plant. Modification can be achieved by methods such as antisense, cosuppression, or over-expression.
By xe2x80x9cplant cell aggregatesxe2x80x9d, xe2x80x9cplant cell linesxe2x80x9d, and xe2x80x9ccallus cell linesxe2x80x9d is meant proliferating masses of tissue composed of a combination of undifferentiated and differentiated cells undergoing de novo morphogenesis and formed by placing a piece of plant material (explant) onto a growth-supporting medium under sterile conditions. The terms xe2x80x9cplant cell aggregatesxe2x80x9d, xe2x80x9cplant cell linesxe2x80x9d, and xe2x80x9ccallus cell linesxe2x80x9d are meant to include Type I and Type II callus cultures in monocotyledonous plants and hypocotyl- and cotyledon derived cultures in dicotyledonous plants. The terms defined herein are not intended to include either plant cell suspension cultures or Type III callus cultures.
By xe2x80x9cplant tissuesxe2x80x9d is meant organized tissues including but not limited to meristems, embryos, pollen, cotyledons, germ cells, and the like.
By xe2x80x9cpromoter regulatory elementxe2x80x9d is meant nucleotide sequence elements within a nucleic acid fragment or gene which controls the expression of that nucleic acid fragment or gene. Promoter sequences provide the recognition for RNA polymerase and other transcriptional factors required for efficient transcription. Promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express sense and antisense gene constructs. Promoter regulatory elements are also meant to include constitutive promoters, tissue-specific promoters, developmental-specific promoters, inducible promoters and the like. Promoter regulatory elements may also include certain enhancer sequence elements that improve transcriptional or translational efficiency.
By xe2x80x9ctissue-specificxe2x80x9d promoter is meant promoter elements responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (i.e., zein, oleosin, napin, ACP, globulin and the like).
By xe2x80x9ctransgenicxe2x80x9d is meant to include any Type I or Type II callus, hypocotyl- or cotyledon derived callus, tissue, plant parts or plants which contains heterologous DNA or a chimeric gene construct that was introduced into said callus, tissue, plant parts or plants by whiskers and was subsequently transferred to later generations by sexual or asexual cell crosses or cell divisions.
By xe2x80x9cwhiskersxe2x80x9d is meant elongated needle-like bodies capable of being produced from numerous substances as described in xe2x80x9cThe Condensed Chemical Dictionary, Seventh Edition, Ed. Arthur and Elizabeth Rose, Reinhold Publishing Corp., New York (1966). The invention is not meant to be limited to the material from which the whiskers are made but instead is meant to define a needle-like shaped structure wherein said whisker is smaller than the cell for which it is intended to be used in the transformation thereof. It is within the scope of this invention for whiskers to be shaped in a manner whereby DNA entry into a cell is facilitated. It is also intended that the scope of said invention include any material having a needle-like shape, said needle-like shaped material being able to perforate a plant cell with or without cell walls and thus facilitate DNA uptake and plant cell transformation. It is also intended that the scope of this invention not include microinjection techniques, such as wherein a DNA molecule is inserted into a cell by passing said DNA through an orifice intrinsic to a needle, said needle being first inserted into said cell. Preferably, whiskers are metal or ceramic needle-like bodies, with those most preferred being made of either silicon carbide or silicon nitride and being 30xc3x970.5 xcexcm to 10xc3x970.3 xcexcm in size.
By xe2x80x9cwhisker-mediated transformationxe2x80x9d is meant the facilitation of DNA insertion into plant cell aggregates and/or plant tissues by whiskers and expression of said DNA in either a transient or stable manner.
In producing plant cell lines, tissues of interest are aseptically isolated and placed onto solid initiation medium whereby processes associated with cell differentiation and specialization occurring in organized plant cell tissues are disrupted, thus resulting in said tissues becoming dedifferentiated. Typically, initiation medium is solidified by adding agar or the like because callus cannot be readily initiated in liquid medium. Media are typically based on the N6 salts of Chu et al., (1978, Proc. Symp. Plant Tissue Culture, Peking Press, p 43-56) being supplemented with sucrose, vitamins, minerals, amino acids, and in some cases, synthetic hormones. However, callus tissues can also proliferate on media derived from the MS salts of Murashige and Skoog, (1962 Physiol. Plant. 15: 473-497). Cultures are generally maintained in a dark, sterile environment at about 28xc2x0 C.
Typically, plant cell lines are preferably derived from tissues found in juvenile leaf basal regions, immature tassels, hypocotyl tissue, and cotylendonary nodes. For maize and rice, plant cell lines which produce meristematic tissue can be used, with those from zygotic embryo tissue being most preferred. Tissues most preferred for producing said plant cell lines are isolated from developing maize ears 10 to 14 days after pollination and non-germinated rice seed. Hypocotyl and cotyledon-derived tissues from seedlings are most preferred for production of cotton plant cell lines.
After placing said tissues on solid medium, new meristems arise after several days to a few weeks from either the scutellar region, in the case of corn and rice, or from hypocotyl or cotyledonary tissue in the case of cotton. These new meristems produce undifferentiated parenchymatous cell aggregates without the structural order characteristic of the tissue from which they are derived. Plant cell aggregates lack any recognizable overall structure and contain only a limited number of the many different kinds of specialized cell types found in intact, organized plant tissues. Said aggregates have been classified into non-embryogenic and embryogenic depending on their regenerative capacity, mode of reproduction, and tissue morphology (Franz, 1988, Ph.D. Thesis, University of Wageningen, The Netherlands).
In corn and rice, non-embryogenic calli are comprised of soft, granular, translucent tissue consisting of elongated, vacuolated cells incapable of plant regeneration. Alternatively, embryogenic, monocotyledonous calli, being capable of somatic embryogenesis and plant regeneration, exist in three distinct morphotypes: Type I; Type II; and Type III.
Type I callus consists of compact, nodular, slow-growing embryogenic callus which proliferates as a mixture of complex tissues exhibiting shoot and/or scutellar-like structures (Phillips et al, 1988, In, Corn and Corn Improvement, pp 345-387). Said callus is characterized by a high degree of cellular differentiation, well developed vascular structures and has been referred to as compact embryogenic callus (U.S. Pat. No. 5,641,664 to Plant Genetic Systems). Essentially all monocotyledonous plants have tissue from which Type I callus can be produced. Plant regeneration in Type I callus normally occurs either through organogenesis by elongation of meristems (Green and Phillips, 1975, Crop Sci., 15:417-421) and/or through somatic embryogenesis from a well defined root-shoot axis (Vasil et al., 1984, Amer. J. Bot. 71:158-161). The origin of regenerated shoots in Type I callus is not always obvious and appears to take place via sub-epidermal meristem formation (Franz and Schel, 1991, Can. J. Bot. 69:26-33).
Type II callus, which is not as common as Type I, consists of soft, friable, embryogenic cells and can only be generated from certain monocotyledonous genotypes (Phillips et al, 1988, In, Corn and Corn Improvement, pp 345-387). It grows rapidly, contains little or no vascular elements and can be described as friable, embryogenic callus (U.S. Pat. No. 5,641,664 to Plant Genetics Systems). A distinguishing feature of Type II callus is that it contains numerous globular somatic embryos attached to suspensor-like structures on its surface through which plant regeneration appears to progress in clearly identifiable stages (Franz, 1988, Ph.D. Thesis, University of Wageningen, The Netherlands).
Type III callus has only been most recently described. Said callus is formed only very rarely, is easily dispersed in liquid and does not have any distinct somatic embryos on its surface. It has also been described as xe2x80x9cfriable, non-mucilaginousxe2x80x9d callus (Shillito et al., 1989, Bio/Technology 7:581-587) and consists predominately of undifferentiated tissues capable of regeneration via somatic embryogenesis. Type III callus is considered the ideal tissue for transformation and regeneration because cells thereof easily disassociate and disperse, and thus, readily form suspension cultures. This type of callus is most rare, distinct from Type II callus, and can only be produced by visually selecting and preferentially enriching for it at each sub-culture passage of Type II cultures (WO94/28148; Zeneca).
In cotton, as with other dicotyledonous plants, callus types are typically not defined as Type I, II, or III. Moreover, hypocotyl- and cotyledon-derived callus cultures from cotton have been classified into non-embryogenic and embryogenic depending on morphology and regenerative capacity (Shoemaker et al., 1986 Plant Cell Rep. 3:178-181). Non-embryogenic callus is comprised of a loose, friable mass of cells that does not exhibit a strong cytoplasm staining reaction and cannot be used to readily regenerate plants. However, embryogenic callus appears as a tightly compact, dense cytoplasmic mass of cells capable of plant regeneration via somatic embryogenesis. Somatic embryos produced therefrom first appear as globular structures which gradually elongate and then begin to exhibit cotyledonary development.
Of the callus types disclosed herein, Type I and Type II callus cultures derived from monocotyledonous plants are preferred in the production and regeneration of plants. Transgenic maize plants generated via whisker-mediated transformation are most preferably made from either Type I or Type II cultures; whereas, Type I callus cultures are most preferred in the production and regeneration of transgenic rice plants. In addition, cotyledonary node derived and hypocotyl-derived cultures are preferred in the production of transgenic cotton plants, with cultures produced from cotyledon tissue being most preferred.
Shoot tips of plants, including maize, rice, and cotton, contain apical meristems where organ primordia form from apical initial and subepidermal cells (Steeves and Sussex, 1989, Patterns in Plant Development, Cambridge University Press). Meristems can be isolated, placed onto shoot multiplication medium, and induced to produce multiple shoots from which plants can be regenerated (Zhong et al., 1992, Planta 187:483-489). Meristems, either freshly isolated or precultured to initiate shoot multiplication, can serve as recipient tissues for whisker-mediated transformation as taught in the present invention. Shoot tips containing meristematic regions are preferably removed from developing embryos (Lowe et al., 1995, Bio/Technology 13:677-682) or germinating seedlings (Gould et al., 1991, Plant Physiol. 95:426-434), transformed using whiskers with or without an osmotic pretreatment, and placed onto shoot proliferation medium containing a selection agent prior to plant regeneration (Zhong et al., 1996, Plant Physiol. 110:1097-1107).
The heterologous DNA used for transformation herein may be circular, linear, double-stranded or single-stranded. Generally, said DNA is a recombinant vector plasmid and contains coding regions therein which serve to promote expression of the heterologous gene of interest as well as provide a selectable marker whereby those tissues containing said gene can be identified. Preferably, these recombinant vectors are capable of stable integration into the plant genome where selection of transformed plant lines is made possible by having said selectable marker expression driven either by constitutive, tissue-specific, or inducible promoters included therein.
One variable present in a heterologous DNA is the choice of the chimeric gene. Chimeric genes, either in the sense or antisense orientation, are expressed in plant cells under control of a constitutive, tissue-specific, developmental, or inducible promoter and the like. Preference for a particular chimeric gene is at the discretion of the artisan; however, chimeric genes can be, but are not limited to, from plants, animals, or bacteria and the like and can used to express proteins either not found in a non-transformed cell or found in a transformed cell. Chimeric genes can be also used for, but are not limited to, up-regulation or down-regulation of an endogenous gene of interest.
Another variable is the choice of a selectable marker. Preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which encode for resistance or tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialophos); imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorsulfuron; bromoxynil, dalapon and the like.
In addition to a selectable marker, it may be desirous to use a reporter gene. In some instances a reporter gene may be used with or without a selectable marker. Reporter genes are genes which are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Weising et al. Ann. Rev. Genetics, 22, 421 (1988), which is incorporated herein by reference. Preferred reporter genes include the beta-glucuronidase (GUS) of the uidA locus of E. coli, the chloramphenicol acetyl transferase gene from Tn9 of E. coli, the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria, and the luciferase genes from firefly Photinus pyralis. An assay for detecting reporter gene expression may then be performed at a suitable time after said gene has been introduced into recipient cells. A preferred such assay entails the use of the gene encoding beta-glucuronidase (GUS) of the uida locus of E. coli as described by Jefferson et al., (1987 Biochem. Soc. Trans. 15, 17-19) to identify transformed cells.
Another variable is a promoter regulatory element. In addition to plant promoter regulatory elements, promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express heterologous genes. For example, promoter regulatory elements of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, see PCT/US96/1682; WO 97/13402 published Apr. 17, 1997) and the like may be used. Plant promoter regulatory elements include but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH promoter, heat-shock promoters and tissue specific promoters.
Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences and the like may be present and thus may improve the transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA by affecting transcription, stability of the mRNA and the like. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include but are not limited to Adh-intron 1, the alfalfa mosaic virus coat protein leader sequence, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan.
Constitutive promoter regulatory elements may also be used thereby directing continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoter regulatory elements are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and may also be used.
Promoter regulatory elements may also be active during a certain stage of the plants"" development as well as active in specific plant tissues and organs. Examples of such include but are not limited to pollen-specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed endosperm specific promoter regulatory elements and the like. Under certain circumstances it may be desirable to use an inducible promoter regulatory element responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; and stress. Other desirable transcription and translation elements functional in plants may also be used. Numerous plant-specific gene transfer vectors are known and available to the skilled artisan.
Heterologous DNA can be introduced into regenerable plant cell cultures via whiskers-mediated transformation. While a general description of the process can be found in U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca, no protocols have been published to date for whisker-mediated transformation of Type I, Type II, hypocotyl or cotyledon regenerable plant cell lines.
In whisker-mediated transformation, DNA uptake into plant material is facilitated by very small, elongated, needle-like particles comprised of a biologically inert material. When said particles are agitated in the presence of DNA and plant cell lines, one or more of the particles produce small punctures in the regenerable plant cell aggregates thereby allowing said aggregates to uptake the DNA. Cells which have taken up the DNA are considered to be transformed. Some transformed cells stably retain the introduced DNA and express it.
The elongated needle-like particles used in plant cell transformation are termed xe2x80x9cwhiskersxe2x80x9d and are preferably made of a high density material such as silicon carbide or silicon nitride; however, any material having a needle-like structure wherein the size of said structure is smaller than the cell intended to be transformed is within the scope of the invention. More preferably, whiskers are made of silicon carbide and are either Silar SC-9 or Alfa Aesar as described herein.
Before transformation, plant cell lines are preferably placed onto anosmotic medium and allowed to incubate thereby enhancing transformation over non-osmotically treated cells. Most preferred is an osmotic medium having therein about 36.4 g/L sorbitol and about 36.4 g/L mannitol. These tissue are maintained on said medium for about 4 h before whisker-mediated transformation. Incubation on medium with osmoticum after transformation at the discretion of the artisan. However, transformation of non-osmotically treated plant cells aggregates and tissues is also within the scope of this invention.
Callus cultures used herein for generation of transgenic plants should generally be about 3 weeks to about 20 weeks old depending on the culture type. Typically, callus should be about midway between transfer periods and thus beyond any lag phase that might be associated with transfer to a new media or before reaching any stationary phase typically associated with a culture being on a plate for an extended period of time. However, plant material can be taken before or after sub-culturing; therefore, harvest timing is not generally believed to be critical to practicing the invention as disclosed herein. The amount of callus tissue used in each transformation can vary with amounts of about 100 mg to about 500 mg preferred, about 100 mg to about 250 mg being more preferred and about 200 mg tissue being most preferred.
For transformation, whiskers are typically placed in a small container, such as a conical or microfuge tube and the like, wherein is placed a mixture comprising the DNA construct of interest, a liquid medium, and callus tissue. The order in which materials are added is not significant to practicing the invention as disclosed herein. Thereafter, the container is sealed and agitated. Unlike particles used in biolistic transformation of plant tissue (Sanford et al., 1990 Physiol. Plantarum, 79:206-209; and U.S. Pat. No. 5,100,712), whiskers do not require any special pretreatment with DNA carriers or precipitants prior to use such as CaCl2, spermidine, sheared salmon sperm DNA and the like.
Agitation time used in the transformation process can vary and is typically from between about 10 sec to about 160 sec. The amount of whiskers added per transformation can also vary from between about 1 mg to about 4 mg per tube. An inverse relationship is observed between the amount of whiskers added and the agitation time needed to obtain optimal transformation. Therefore, the amount of whiskers added and the agitation time needed to achieve transformation is determinable by one having skill in the art. In addition, the volume of liquid medium added can vary from about 200 xcexcL to about 1000 xcexcL, with about 200 xcexcL being preferred. Moreover, the amount of heterologous DNA added can vary from a preferred amount of about 10 xcexcL to about 100 xcexcL of 1 mg/mL solution. The volume of DNA added is not as critical of factor to the invention as disclosed herein as the final DNA concentration. However, preferred final DNA concentrations are from about 0.03 xcexcg/xcexcL to about 0.14 xcexcg/xcexcL. The scope of the present invention is not intended to be limited to said container size, the amount or concentration of heterologous DNA added, the volume of heterologous DNA added, the amount of the liquid medium added, the amount of callus material added or the amount of whiskers added as disclosed herein. The scope of the invention is also not intended to be limited by the instrumentation used to agitate the mixture or whether agitation is accomplished by manual or mechanical means.
Once the plant cell lines have been perforated and the heterologous DNA has entered therein, it is necessary to identify, propagate, and select those cells which not only contain the heterologous DNA of interest but are also capable of regeneration. Said cells and plants regenerated therefrom can be screened for the presence or absence of the heterologous DNA by various standard methods including but not limited to assessment of reporter gene expression. Alternatively, transmission of a selectable marker gene along with or as part of the heterologous DNA allows those cells containing said DNA to be identified by use of a selective agent.
Selection of only those cells containing and expressing the heterologous DNA of interest is a critical step in production of fertile, transgenic plants. Selection conditions must be chosen in such a manner as to allow growth of transformed cells while inhibiting growth of untransformed cells, which initially, are far more abundant. In addition, selection conditions must not be so severe as to cause transformed cells to lose their plant regenerability, future viability or fertility. A skilled artisan can easily determine appropriate conditions for selecting transformed cells expressing a particular selectable marker by performing growth inhibition curves. Growth inhibition curves are generated by plotting cell growth versus selective agent concentration. Typically, selective agent concentrations are set at a concentration whereby almost all non-transformed cells are growth inhibited but yet are not killed. Preferred are selective agent concentrations wherein 90-99% of non-transformed cells are growth inhibited but yet not killed. Most preferred are selective agent concentrations wherein 97-99% of non-transformed cells are growth inhibited but yet not killed.
Transformed callus tissues transferred and exposed to selective agents are generally incubated on solid medium supportive of growth. The medium preferred for each type of tissue has been well defined in the art. After initial exposure to selective agents, tissues are transferred periodically to fresh medium while maintaining selective agent concentrations. After transformed cell mass has essentially doubled in size, masses showing the most growth and appearing to be healthy are selected and transferred to fresh medium having selective agent concentrations wherein non-transformed cells will be killed. Repeated selection and transference of growing cells to fresh medium result eventually in a selected group of cells comprised almost exclusively of transformed cells containing the heterologous DNA of interest.
Regeneration, while important to the present invention, may be performed in any conventional manner available to the skilled artisan. If cells have been transformed with selectable marker gene, the selective agent may be incorporated into the regeneration media to further confirm that the regenerated plantlets are transformed. After subsequent weeks of culturing, regenerated plantlet immune to the selective agent can be transferred to soil and grown to maturity.
Callus and plant derived therefrom can be identified as transformants by phenotypic and/or genotypic analysis. For example, if an enzyme or protein is encoded by the heterologous DNA, enzymatic or immunological assays specific for the particular enzyme or protein can be used. Other gene products may be assayed by using suitable bioassays or chemical assays. Other techniques include analyzing the genomic component of the plant using methods as described by Southern ((1975) J. Mol. Biol., 98:503-517), polymerase chain reaction (PCR) and the like.
Plants regenerated from transformed callus are referred to as the RO generation or RO plants. Seed produced by various sexual crosses from plants of this generation are referred to as R1 progeny. R1 seed are then germinated to produce R1 plants. Successful transmission and inheritance of heterologous DNA to R1 plants and beyond should be confirmed using the methods described herein.
Generally, the commercial value of transformed corn and progeny thereof will be of greatest value if the heterologous DNA can be incorporated into many different hybrids. This may be achieved by incorporating the heterologous DNA into a large number of parental lines directly as described herein by creating plant cell aggregates of said lines and transforming said lines with whiskers. In addition, this may also be accomplished by crossing initial transgenic fertile plants to normal elite inbred lines and then crossing the progeny thereof back to the normal parent. Progeny from this cross will segregate such that some plants will contain the heterologous DNA of interest and some plants will not. Crossing of lines is continued until the original normal parent has been converted to a genetically modified line containing the heterologous DNA of interest and also possessing essentially all attributes associated with that line originally. Corn breeding techniques needed to accomplish elite germplasm lines and inbreds thereof are well known to the skilled artisan.